Binding partners of the placental growth factor, especially antibodies directed against the placental growth factor, and production and use thereof

ABSTRACT

The invention relates to binding partners of the placental growth factor (or placenta growth factor, PIGF), especially antibodies directed against the placental growth factor, and production and use thereof.

PRIOR APPLICATIONS

This application a continuation of application Ser. No. 11/920,160,filed Nov. 9, 2007, which is a 35 U.S.C. §371 National Phase Entryapplication of International Application PCT/EP2006/004278, filed May 8,2006, which designated the U.S. and which claims the benefit under 35U.S.C. §119 of German Application No. 10 2005 022 047.9, filed May 9,2005, the entire contents of which are hereby incorporated by reference.

FIELD OF THE INVENTION

The invention relates to binding partners of the placental growth factor(or placenta growth factor, PIGF), especially antibodies directedagainst the placental growth factor, and production and use thereof.

BACKGROUND OF THE INVENTION

PIGF is involved in important physiological and pathological processes,in particular angiogenesis. It plays an important part in tumorprogression, kidney diseases, which are caused in particular by diabetesmellitus, in psoriasis, inflammatory diseases, in particular rheumatoidarthritis, in cardiovascular diseases and the like [Iyer, S.; Leonidas,D. D.; Swaminathan, G. J.; Maglione, D.; Battisti, M.; Tucci, M.;Persico, M. G.; Acharya, K. R. J Biol Chem 2001, 276, (15),12153-61./Iyer, S.; Acharya, K. R. Trends Cardiovasc Med 2002, 12, (3),128-34./Heeschen, C.; Dimmeler, S.; Fichtlscherer, S.; Hamm, C. W.;Berger, J.; Simoons, M. L.; Zeiher, A. M. JAMA 2004, 291, (4),435-41./Yang, W.; Ahn, H.; Hinrichs, M.; Torry, R. J.; Torry, D. S. JReprod Immunol 2003, 60, (1), 53-60.].

PIGF is mainly expressed in the placenta and belongs to the“cysteine-knot” protein family. PIGF occurs in different forms.Different forms of PIGF are (I) primary isoforms and (II) secondaryisoforms. (III) In addition, a distinction can be made between free PIGF(fPIGF) and bound PIGF (gPIGF).

(I) Primary PIGF Isoforms

Primary PIGF isoforms are characterized by the primary sequence, i.e.the order of the amino acids in the protein. Alternative splicing andposttranslational modifications, such as glycosylations,phosphorylations, degradation (degradation products, fragments, etc.),acetylations etc., lead to different primary PIGF isoforms. To date,four different primary isoforms of human PIGF, PIGF-1 (PIGF-131), PIGF-2(PIGF152), PIGF-3 (PIGF-203) and PIGF-4 have been described.

The sequence of the PIGF-1 precursor (Sequence number (SN) 1V) is asfollows:

SN 1V (SEQ ID NO: 1) 1

61 VVSEYPSEVE HMFSPSCVSL LRCTGCCGDE NLHCVPVETA NVTMQLLKIR SGDRPSYVEL 121

Secreted PIGF-1 does not as a rule possess the leader sequence of thePIGF-1 precursor (PIGF precursor) and thus begins at the N-terminus withalanine (A) (stated as

in the sequence of the PIGF-1 precursor, see above). This as a ruleapplies also to the other primary PIGF isoforms.

The sequence of the primary PIGF-1 isoform is thus as follows:

SN 1: (SEQ ID NO: 2) 1

61

121 KPERCGDAVP RR

In this primary sequence, possible sites for post-translationalmodifications, and hence also the presence of posttranslationallymodified primary isoforms, can be discerned. For example, in general invivo the posttranslationally modified primary PIGF isoform of PIGF-1glycosylated at position 84 (asparagine,

) is present.

As the first N-terminal amino acid of the primary isoform PIGF-1,methionine (M) is often stated instead of alanine. This in generalrelates to recombinantly, for example in Escherichia coli (E. coli),expressed PIGF-1(rPIGF-1), and in particular to the human rPIGF-1(rhPIGF-1). Here AUG, which codes for methionine, is used as the startcodon. Such a PIGF expressed in E. coli has no posttranslationalmodifications, in particular also no glycosylations.

The sequence of the recombinant, human, primary PIGF-1 isoform isgenerally stated as follows:

SN 1RH: (SEQ ID NO: 3) 1

61 VSLLRCTGCC GDENLHCVPV ETANVTMQLL KIRSGDRPSY VELTFSQHVR CECRPLREKM 121KPERCGDAVP RR

Through alternative splicing, the sequence RRRPKGRGKRRREKQRPTDCHL (SEQID NO: 64) is present in the PIGF-2 isoform instead of the arginine (R)124. The sequence of the primary PIGF-2 isoform thus reads:

SN 2: (SEQ ID NO: 4) 1 ALPAVPPQQW ALSAGNGSSE VEVVPFQEVW GRSYCRALERLVDVVSEYPS EVEHMFSPSC 61 VSLLRCTGCC GDENLHCVPV ETANVTMQLL KIRSGDRPSYVELTFSQHVR CECRPLREKM 121 KPERRRPKGR GKRRREKQRP TDCHLCGDAV PRR

An insert of 72 amino acids inserted by alternative splicing(HSPGRQSPDMPGDFRADAPSFLPPRRSLPMLFRMEWGCALTGSQSAVWPSSPVPEEIPRMHPGRNGKKQQRK (SEQ ID NO: 65)) leads to the sequence ofthe primary PIGF-3 isoform:

SN 3: (SEQ ID NO: 5) 1ALPAVPPQQW ALSAGNGSSE VEVVPFQEVW GRSYCRALER LVDVVSEYPS EVEHMFSPSC 61

121

181

The primary PIGF-4 isoform contains sequences both of the PIGF-2 isoform(italic) and also of the PIGF-3 isoform (underlined):

SN 4: (SEQ ID NO: 6) 1ALPAVPPQQW ALSAGNGSSE VEVVPFQEVW GRSYCRALER LVDVVSEYPS EVEHMFSPSC 61

121

181

(II) Secondary PIGF Isoforms

Secondary PIGF isoforms result from the combination of primary PIGFisoforms or other molecules, in particular molecules which arehomologous to PIGF. The primary PIGF isoforms or other molecules aresubunits of the secondary PIGF isoforms. In general, secondary PIGFisoforms consist of two subunits. Thus PIGF is as a rule present as adimer, i.e. as a homodimer or a heterodimer. Homodimers consist of twoidentical primary PIGF isoforms (subunits) such as PIGF-1×PIGF-1,PIGF-2×PIGF-2, PIGF-3×PIGF-3 and PIGF-4×PIGF-4. Heterodimers consist oftwo different primary PIGF isoforms or of one primary PIGF isoform andone other molecule, in particular a PIGF homolog such as vascularendothelial growth factor (VEGF) and primary isoforms thereof. Possibleexamples of heterodimers are PIGF-1×PIGF-2, PIGF-3×PIGF-4, PIGF-1×VEGF,etc.

(III) Free PIGF (fPIGF) and Bound PIGF (gPIGF)

Since PIGF forms complexes with binding partners, the complexed or boundforms of PIGF must also be considered as well as the isoforms. Inprinciple, the free primary, but in particular the free secondary PIGFisoforms (free PIGF, fPIGF), should be distinguished from the complexedor bound forms (bound PIGF, gPIGF). gPIGF is for example homodimericPIGF-1 which is present in complexed form. These can be simplecomplexes, i.e. a PIGF-1 homodimer is bound to a receptor, for examplethe membrane-bound fms-like tyrosine kinase receptor-1 (mFlt-1). Otherexamples are complexes with the soluble Flt-1 (sFlt-1), withneurophilins (NP; in particular NP-1 and NP-2), with the kinasedomain-containing receptor/fetal liver kinase receptor (KDR/Flk-1,VEGFR-2), with heparin sulfate proteoglycans (HSPG) and isoforms,homologs, fragments and degradation products thereof. Multilayerconstituted complexes of several and sometimes different PIGF isoformsand several and sometimes different binding partners, in particularreceptors, are also possible.

The function of PIGF is mediated, modulated or inhibited by binding tothe membrane-bound or soluble fms-like tyrosine kinase receptor-1(fms-like tyrosine kinase receptor-1 (Flt-1) or Vascular EndothelialGrowth Factor (VEGF) receptor-1 (VEGFR-1)) and the kinasedomain-containing receptor/fetal liver kinase receptor (KDR/Flk-1 orVEGFR-2). In addition to other possible functions of PIGF, the bindingof PIGF to membrane-bound Flt-1 (mFlt-1) is especially important. Thisresults in mFlt-1 transphosphorylation and thus activates signaltransduction cascades [Iyer, S.; Acharya, K. R. Trends Cardiovasc Med2002, 12, (3), 128-34.].

In contrast to this, it is presumed that the binding of PIGF to solubleFlt-1 (sFlt-1) serves to reduce the physiological activity of PIGF[Iyer, S.; Acharya, K. R. Trends Cardiovasc Med 2002, 12, (3), 128-34.].Furthermore it is presumed that the PIGF isoform is involved. PIGF-2,which is possibly linked to the membrane, has a cationic insert of 21amino acids at the carboxy terminal end. Through the binding of anionic,in particular polyanionic, substances such as heparin, heparin sulfateproteoglycans, etc., further functions can be mediated. TheN-glycosylation of asparagine (Asn) 84 and the amino acid sequence whichis present in PIGF-3 can also have similar effects. Moreover, it ispresumed that the binding of PIGF and VEGF has yet another effect, sincehere the VEGF expression and thus its activity is negatively regulated[Iyer, S.; Acharya, K. R. Trends Cardiovasc Med 2002, 12, (3), 128-34.].In summary, this means that the various forms of PIGF have differentfunctions or exert different effects.

For the current detection methods and binding partners, in particularantibodies, which are at present used for analytical and diagnosticpurposes, there is the problem that the different forms of PIGF are not,or not efficiently enough (not sufficiently specifically) distinguished.For example, the “Anti-human PIGF Antibody” from R&D Systems, Inc. doesnot exclusively recognize certain PIGF forms, in particular rhPIGF-1homodimer but also the heterodimer of rhPIGF and VEGF and rhPIGF-2 (R&DSystems Catalog Number: AF-264-PB or DPG00 product descriptions).

Furthermore, fPIGF or gPIGF are not exclusively detected, i.e. with theexisting antibodies, a distinction between fPIGF and gPIGF is not or notsufficiently efficiently made. In particular, the specific detection offPIGF is inadequate. This is demonstrated by the fact that rhFlt-1 inthe form of rhFlt-1/Fc has an effect on the determination of PIGF (R&DSystems Catalog Number: DPG00). This non-specificity is confirmed in theliterature [Maynard, S. E.; Min, J. Y.; Merchan, J.; Lim, K. H.; Li, J.;Mondal, S.; Libermann, T. A.; Morgan, J. P.; Sellke, F. W.; Stillman, I.E.; Epstein, F. H.; Sukhatme, V. P.; Karumanchi, S. A. J Clin Invest2003, 111, (5), 649-58.]. Maynard et al. show that the relevant R&DSystems ELISA (R&D Systems Catalog Number: AF-264-PB or DPG00) does showa certain specificity for fPIGF, however the studies performed show thatthis specificity is low. In the determination of 0.5 ng/mL rhPIGF-1, asignal reduction of only about 12% is to be seen in the presence of 0.5ng/mL sFlt-1. Even with a 10-fold excess of sFlt-1 (5 ng/mL), there wasonly a signal reduction by the factor of 2. A more pronounced signalreduction would occur with higher specificity of the antibodies usedtowards fPIGF.

SUMMARY OF THE INVENTION

The purpose of the present invention was thus to provide processes orcomponents which enable the specific detection of particular PIGF forms,in particular by means of specific binding partners, in particularantibodies.

The solution of this problem consists in the provision of the objectsand processes according to the invention described in the claims.

In particular, the purpose is achieved by the provision of bindingpartners, in particular antibodies, which bind specifically to theprimary isoforms of PIGF. These binding partners, in particularantibodies, form the basis for the immunological detection and thequantification of the primary and secondary isoforms and of the free orbound PIGF forms. This applies in particular to biological materials, inparticular plasma samples, for diagnostic applications. Therapeuticapplications are likewise possible.

Surprisingly, the different forms of PIGF can be detected as describedbelow. The selection of binding partners and of substances for thepreparation of specific binding partners for the specific detection offree PIGF is described in more detail below:

According to the invention, specific binding partners, in particularantibodies and proteins of the receptor tyrosine kinase family, inparticular Flt-1, Flt-2, Flt-3, Flt-4, preferably Flt-1, homologs,fragments and degradation products, which bind in the region of thereceptor-binding domains at the poles of the secondary PIGF isoforms areespecially suitable for the detection of fPIGF, in particular ofnon-m/sFlt-1-bound PIGF.

Especially suitable are specific binding partners, in particularantibodies which are prepared with the use of free primary and secondaryPIGF isoforms, in particular secondary PIGF isoforms, preferably PIGFhomodimers, particularly preferably PIGF-1 homodimer, in particularrhPIGF-1 homodimer, preferably N-glycosylated rhPIGF-1 homodimer, forexample by immunizations, and which on characterization show thatinteractions in the region of the receptor binding site are entered intoor are necessary during binding.

The “head-to-tail” orientation of the monomers (primary PIGF isoforms)in dimers (secondary PIGF isoforms) has the effect that eachreceptor-binding domain is situated at the poles of the PIGF dimers. Thereceptor binding takes place at the monomer-monomer boundary and notexclusively on one monomer.

In particular, surprisingly, peptides which only possess the sequenceinformation of one monomer and hence do not include the wholereceptor-binding domain, which is made up of both monomers, areespecially suitable as immunization antigens for the production offPIGF-specific antibodies.

Peptides which contain amino acids which are important for receptorinteractions or peptides which cover sequence regions in the vicinitythereof are especially suitable as immunization antigens. The aminoacids which are particularly but not exclusively important for the Flt-1receptor interactions are shown underlined below within the PIGF-1sequence. The PIGF-1 sequence was selected by way of example forillustration. These amino acids are also important for receptorinteractions in the case of the other PIGF isoforms. The following aminoacid is disclosed as SEQ ID NO: 2.

SN 1: (SEQ ID NO: 2) 1

61

121 KPERCGDAVP RR

E-112 and P-115, but in particular P-1I5, which probably play asecondary or indeed no part in the corresponding receptor interactionsin the case of PIGF-3 and PIGF-4, represent an exception, since theinsert of 72 amino acids (SN 3 and SN 4) described above lies betweenthese amino acids.

The following peptides are especially suitable as immunization antigensfor the preparation of fPIGF-specific antibodies (Immunization antigennumbers (IAN) 1-4):

IAN 1: (SEQ ID NO: 7) SAGNGSSEVE VVPFQEVWGR SYCRALERLV IAN 2:(SEQ ID NO: 26) LRCTGCCGDE NLHCVPVET IAN 3: (SEQ ID NO: 33)VETANVTMQL LKIRSGDRP SYVELTFSQH IAN 4: (SEQ ID NO: 66)TFSQHVRCEC RPLREKMKPE RCGDAVPRR

Immunization antigens can also contain sequences from different peptideswith the immunization antigen numbers IAN 1-4. For example, thefollowing peptide can be used:

IAN 2/3: (SEQ ID NO: 43) VPVETANVTM QL

Particular embodiments are explained in more detail below.

DETAILED DESCRIPTION OF THE INVENTION

An object of this invention are peptides consisting of 4 to 30 aminoacids, preferably 5 to 20 amino acids, quite particularly preferably 10to 15 amino acids, which are characterized in that they contain theamino acid sequences FQEVWGRSY (IAN: 1-1) (SEQ ID NO: 8), SAGNGSSEVEVV(IAN: 1-1-1) (SEQ ID NO: 9), VVPFQEVWGRSY (IAN: 1-1-2) (SEQ ID NO: 11),GDENL (IAN: 2-1) (SEQ ID NO: 27), GCCGDENLH (IAN: 2-1-1) (SEQ ID NO:28), QLLKIRSGDRPSY (IAN: 3-1) (SEQ ID NO: 34), QLLKI (IAN: 3-2) (SEQ IDNO: 35), RPSYV (IAN: 3-3) (SEQ ID NO: 36), RSGDRPSYVELT (IAN: 3-3-1)(SEQ ID NO: 37) and/or ECRP (IAN: 4-1) (SEQ ID NO: 46).

Peptides which contain 5 consecutive amino acids of the statedsequences, i.e. for example EVVPF (IAN: 1-2) (SEQ ID NO: 13), VVPFQ(IAN: 1-3) (SEQ ID NO: 14), VPFQE (IAN: 1-4) (SEQ ID NO: 15), PFQEV(IAN: 1-5) (SEQ ID NO: 16), FQEVW (IAN: 1-6) (SEQ ID NO: 17), QEVWG(IAN: 1-7) (SEQ ID NO: 18), EVWGR (IAN: 1-8) (SEQ ID NO: 19), VWGRS(IAN: 1-9) (SEQ ID NO: 20), WGRSY (IAN: 1-10) (SEQ ID NO: 21), GRSYC(IAN: 1-11) (SEQ ID NO: 22), RSYCR (IAN: 1-12) (SEQ ID NO: 23), SYCRA(IAN: 1-13) (SEQ ID NO: 24), YCRAL (IAN: 1-14) (SEQ ID NO: 25) or GCCGD(IAN: 2-2) (SEQ ID NO: 30), CCGDE (IAN: 2-3) (SEQ ID NO: 31), CGDEN(IAN: 2-4) (SEQ ID NO: 32), etc. down to PLREK (IAN: 4-2) (SEQ ID NO:47), are also particularly preferable.

Immunization antigens can be used for the immunization unbound and/orcarrier-bound. In order to facilitate coupling to typical carriers, forexample proteins, such as ovalbumin, albumin or keyhole limpethemocyanin, peptides which contain a lysine are preferably synthesized.The following peptides are particularly suitable for this: SAGNGSSEVEVVK(IAN: 1-1-1K) (SEQ ID NO: 10), SGDRPSYVELTK (IAN: 3-3-1K) (SEQ ID NO:38), VPVETANVTMQLK (IAN: 2/3K) (SEQ ID NO: 44), VVPFQEVWGRSYK (IAN:1-1-2K) (SEQ ID NO: 12) and GCCGDENLHK (IAN: 2-1-1K) (SEQ ID NO: 29).

“Multiple antigenic peptide systems” can also be used as immunizationantigens [Tam, J. P. Proc Natl Acad Sci USA 1988, 85, 5409-5413]. Inparticular, 8-mers of IAN 1-1-1, IAN 3-3-1 or IAN 2/3 can be used:

IAN 1-1-1\8-mer: (SEQ ID NO: 67) (SAGNGSSEVEVV)₈K₄K₂K-βAIAN 3-3-1\8-mer: (SEQ ID NO: 68) (RSGDRPSYVELT)₈K₄K₂K-βA IAN 2/3\8-mer:(SEQ ID NO: 69) (VPVETANVTMQL)₈K₄K₂K-βA

The selection of substances for the preparation of specific bindingpartners for the specific detection of gPIGF is explained in more detailbelow:

With the use of gPIGF, specific binding partners, in particularantibodies, are identified or prepared, for example by immunizations.These specific binding partners are distinguished in theircharacterization in that, during binding, interactions occur both withthe PIGF and also with the bound binding partner.

For example, for the preparation of antibodies PIGF, which forms acomplex with a binding partner, can be used in the immunization. Inparticular, these can be PIGF-1 homodimers which are complexed withsFlt-1. Corresponding complexes consisting of the relevant homologs,fragments, etc. can also be used.

The selection of substances for the preparation of specific bindingpartners for the specific detection of posttranslationally modified, inparticular glycosylated PIGF is described in more detail below.

For this, PIGF or corresponding peptides with or withoutposttranslational modification are used for the preparation of specificbinding partners, in particular antibodies. The specific bindingpartners are then suitable for specifically detecting the presence orabsence of posttranslational modifications.

For example, N-glycosylated peptides which contain the sequenceVETANVTMQ (IAN: 3-4) (SEQ ID NO: 39) or parts thereof, for example VETAN(IAN: 3-5) (SEQ ID NO: 40), TANVT (IAN: 3-6) (SEQ ID NO: 41) or NVTMQ(IAN: 3-7) (SEQ ID NO: 42), can be used for the preparation of specificbinding partners, in particular antibodies, which can be usedspecifically for the detection of PIGF glycosylated on asparagine 84 (N84). As well as these N-glycosylated peptides, it is also possible touse glycosylated PIGF, or corresponding fragments.

In addition, through the use of appropriate homologous, non-glycosylatedpeptides, PIGF and appropriate fragments, binding partners can beprepared which can be used in the specific detection of non-glycosylatedPIGF.

The selection of substances for the preparation of specific bindingpartners for the specific detection of PIGF-2 is explained in moredetail below:

Here, primary or secondary PIGF-2 isoforms, fragments thereof orcorresponding peptides are used for the preparation of specific bindingpartners, in particular antibodies. Peptides and fragments thereof whichcontain the following sequence or parts thereof are especially suitableas immunization antigens for the preparation of PIGF-2-specificantibodies:

(SEQ ID NO: 48) IAN 5:

Particularly preferably, peptides which contain the underlined sequenceor parts thereof should be used.

Peptides which contain 5 consecutive amino acids of the sequence statedabove (IAN 5) are particularly suitable, for example MKPER (IAN: 5-1)(SEQ ID NO: 49), KPERR (IAN: 5-2) (SEQ ID NO: 50), etc. down to LCGDA(IAN: 5-3) (SEQ ID NO: 51).

As well as the specific binding partners for PIGF-2, in particularantibodies, which are generated by immunization or other procedures withthe proteins and peptides described above, it is also possible to usespecific binding partners such as anionic compounds, in particularpolyanionic compounds, preferably heparin compounds, in particularheparin sulfate proteoglycans. In a further embodiment, proteins whichbelong to the family of the semaphorin receptors, in particularneuropilins, preferably neuropilin-1 (NP-1) and neuropilin-2 (NP-2) areused as specific PIGF-2 binding partners.

Below, the selection of substances for the preparation of specificbinding partners for the specific detection of PIGF-3 is described inmore detail:

Here, primary or secondary PIGF-2 isoforms, fragments thereof orcorresponding peptides are used for the preparation of specific bindingpartners, in particular antibodies. Peptides and fragments thereof whichcontain the following sequence or parts thereof are especially suitableas immunization antigens for the preparation of PIGF-3-specificantibodies:

IAN 6: (SEQ ID NO: 52) 1

51

Particularly preferably, peptides which contain the underlined sequenceor parts thereof should be used.

Peptides which contain 5 consecutive amino acids of the stated sequenceIAN 6, i.e. for example CECRH (IAN: 6-1) (SEQ ID NO: 53), ECRHS (IAN:6-2) (SEQ ID NO: 54) etc. down to KPLRE (IAN: 6-3) (SEQ ID NO: 55), areparticularly suitable.

The selection of binding partners and of substances for the preparationof specific binding partners for the specific detection of PIGF-4 isdescribed in more detail below:

Here, primary or secondary PIGF-4 isoforms, fragments thereof orcorresponding peptides are used for the preparation of specific bindingpartners, in particular antibodies. Peptides and fragments thereof whichcontain the following sequence or parts thereof are especially suitableas immunization antigens for the preparation of PIGF-4-specificantibodies:

(SEQ ID NO: 56) IAN 7:

Particularly preferably, peptides which contain the underlined aminoacids should be used.

Peptides which contain the following sequences are especially suitable:QQRKP (IAN: 7-1) (SEQ ID NO: 57), QRKPL (IAN: 7-2) (SEQ ID NO: 58),RKPLR (IAN: 7-3) (SEQ ID NO: 59), KPLRE (IAN: 6-3) (SEQ ID NO: 55),MKPER (IAN: 5-1) (SEQ ID NO: 49), KPERR (IAN: 5-2) (SEQ ID NO: 50),PERRR (IAN: 7-4) (SEQ ID NO: 60) and ERRRP (IAN: 7-5) (SEQ ID NO: 61).

Since PIGF-4 contains both specific PIGF-2 sequences and also specificPIGF-3 sequences, the detection of PIGF-4 can be carried out by means ofspecific PIGF-2 binding partners and PIGF-3 binding partners accordingto the invention, in particular antibodies which are prepared by meansof antigens according to the invention, in particular peptides.

The selection of binding partners and of substances for the preparationof specific binding partners for the specific detection of PIGF/VEGFheterodimers is described in more detail below:

Since PIGF/VEGF heterodimer contains both specific PIGF sequences (PIGF1-4) and also specific VEGF sequences (VEGF isoforms), the detection ofPIGF/VEGF hetero-dimers can be carried out by means of specific PIGFbinding partners according to the invention, in particular antibodies,which are prepared by means of antigens according to the invention, inparticular peptides, and VEGF antibodies.

In addition, according to the invention the VEGF/PIGFheterodimer-specific binding partner KDR/Flk-1, and isoforms, homologs,fragments and degradation products thereof may be used.

In particular, specific binding partners, especially antibodies, whichwere identified or prepared with the use of VEGF/PIGF heterodimers, forexample by immunizations, and which during characterization show thatduring binding interactions both with the VEGF monomer and also with thePIGF monomer are entered into, can also be used.

A preferred process for the preparation of the peptides according to theinvention, which inter alia are used as immunization antigens, is solidphase synthesis, wherein a multiple copy number of a peptide issynthesized on a lysine core [see also Tam J. P. (1988) Proc. Natl.Acad. Sci. USA 85: 5409-5413]. The peptide synthesis is preferablyperformed in accordance with a standard protocol by means of automaticmachines, such as are for example supplied by Applied Biosystems (USA).Such multimeric peptides can moreover be bound to a carrier protein.

The specific binding partners according to the invention bind to anepitope. A “specific binding partner” should be understood to mean amember of a specific binding pair. The members of a specific bindingpair are two molecules which each have at least one structurecomplementary to a structure of the other molecule, whereby the twomolecules are capable of binding via a binding of the complementarystructures. The term molecule also includes molecule complexes such asfor example enzymes which consist of apo- and coenzyme, proteins whichconsist of several subunits, lipoproteins consisting of protein andlipids, etc. Specific binding partners can be naturally occurring butalso substances prepared for example by chemical synthesis,microbiological techniques and/or genetic engineering processes. Thefollowing list serves for the illustration of the term specific bindingpartners, without however limiting this to these substances:thyroxine-binding globulin, steroid-binding proteins, antibodies,antibody fragments, designed repeat proteins, protein scaffolds,ankyrins, leucine-rich repeats, anticalins, duocalins, lipocalins,Affi-Bodies®, antigens, haptens, enzymes, lectins, nucleic acids, inparticular aptamers, repressors, oligo- and polynucleotides, protein A,protein G, avidin, streptavidin, biotin, complement component Clq,nucleic acid-binding proteins, etc. Examples of specific binding pairsare: antibody-antigen, antibody-hapten, operator-repressor,nuclease-nucleotide, biotin-avidin, lectin-polysaccharide,steroid-steroid-binding protein, active substance-active substancereceptor, hormone-hormone receptor, enzyme-substrate, IgG-protein A,complementary oligo- or polynucleotides, etc.

The term “peptide” in the sense of this invention includes acid amideswhich on hydrolysis decompose into amino acids, for example amino acidpolymers such as for example polypeptides, oligopeptides, proteins orprotein fragments.

The peptides according to the invention can be used as immunizationantigens for the preparation of the antibodies according to theinvention or also for affinity chromatographic purification of theantibodies according to the invention. Further, the peptides accordingto the invention can also be used in a process for the quantitative orqualitative detection of an analyte, preferably of the various PIGFforms. The peptides according to the invention can also be linked to asolid phase and/or a component of a signal-generating system, forexample in an immunoassay.

The term “antigens” includes monovalent and polyvalent antigens. Apolyvalent antigen is a molecule or a molecule complex onto which morethan one immuno-globulin can simultaneously bind, while with amonovalent antigen only a single antibody can bind at the same time. Amolecule which in itself alone is not immunogenic, but which is normallybound to a carrier for immunization purposes is usually described as ahapten.

The term “antibody” in the sense of this invention should be understoodto mean an immunoglobulin, for example an immunoglobulin of the class orsubclass IgA, IgD, IgE, IgG1, IgG2a, IgG2b, IgG3, IgG4 or IgM. Anantibody has at least one binding site (often called a paratope) for oneepitope (often also called an antigenic determinant) on an antigen orhapten. Such an epitope is for example characterized by its spatialstructure and/or by the presence of polar and/or apolar groups. Thebinding site of the antibody is complementary to the epitope. Theantigen-antibody reaction or the hapten-antibody reaction functionsaccording to the so-called “lock and key” principle and is as a rulehighly specific, i.e. the antibodies are capable of distinguishing smalldeviations in the primary structure, in the charge, in the spatialconfiguration and the steric arrangement of the antigen or hapten. Inparticular, the so-called “complement-arity determining regions” of theantibody contribute to the binding of the antibody to the antigen orhapten.

The term “antibody” in the sense of this invention should however beunderstood to mean not only complete antibodies, but expressly alsoantibody fragments, such as for example Fab, Fv, F(ab′)2, Fab′; and alsochimeric, humanized, bi- or oligospecific, or “single chain” antibodies;and also aggregates, polymers and conjugates of immunoglobulins and/orfragments thereof, provided that the binding properties to the antigenor hapten are retained. Antibody fragments can for example be preparedby enzymatic cleavage of antibodies with enzymes such as pepsin orpapain. Antibody aggregates, polymers and conjugates can be generated bya variety of methods, for example by heat treatment, reaction withsubstances such as glutaraldehyde, reaction with immunoglobulin-bindingmolecules, biotinylation of antibodies followed by reaction withstreptavidin or avidin, etc.

An antibody in the sense of this invention can be a monoclonal or apolyclonal antibody. The antibody can be prepared by the usual process,for example by immunization of a human or an animal, such as for examplemouse, rat, guinea pig, rabbit, horse, donkey, sheep, goat or chicken[see also Messerschmid (1996) BIOforum 11: 500-502], followed byisolation of the antiserum; or by establishing hybridoma cells followedby purification of the secreted antibody; or by cloning and expressionof the nucleotide sequences or modified versions thereof, which encodethe amino acid sequences which are responsible for the binding of thenatural antibody to the antigen and/or hapten.

Antibodies according to the invention are in particular those antibodieswhich bind to the proteins, protein complexes or peptides describedabove.

Through the provision of the antibodies according to the invention it isnow possible for the person skilled in the art, for example bycompetition experiments [see also Peters et al. (1985) MonoklonaleAntikörper, Springer Verlag, Chapter 12.2 “Epitop-Analyse”], to identifyother specific binding partners, antibodies expressly includedtherewith, which bind to the epitope of an antibody according to theinvention. Thus specific binding partners can now be selected by meansof phage display libraries, with synthetic peptide databases or by meansof “recombinatorial antibody libraries” [Larrick & Fry (1991) HumanAntibodies and Hybridomas 2: 172-189].

Also an object of this invention is an antibody according to theinvention which is linked to a solid phase and/or a component of asignal-forming system.

The term “solid phase” in the sense of this invention includes an objectwhich consists of porous and/or nonporous, as a rule water-insolublematerial and which can take a great variety of forms, such as forexample that of vessels, tubes, microtitration plates, spheres,microparticles, rods, strips, filter or chromatography paper, etc. As arule the surface of the solid phase is hydrophilic or can be madehydrophilic. The solid phase can consist of a great variety of materialssuch as for example inorganic and/or organic materials, synthetic,naturally occurring and/or modified naturally occurring materials.Examples of solid phase materials are polymers, such as for examplecellulose, nitrocellulose, cellulose acetate, polyvinyl chloride,polyacrylamide, crosslinked dextran molecules, agarose, polystyrene,polyethylene, polypropylene, poly-methacrylate or nylon; ceramics,glass, metals, in particular noble metals such as gold and silver;magnetite; mixtures or combinations of the same, etc. Cells, liposomesor phospholipid vesicles are also covered by the term solid phase.

The solid phase can have a coating of one or more layers, for example ofproteins, carbohydrates, lipophilic substances, biopolymers, organicpolymers or mixtures thereof, in order for example to suppress orprevent the nonspecific binding of sample components to the solid phaseor for example to achieve improvements in the suspension stability ofparticulate solid phases, storage stability, dimensional stability orresistance to UV light, microbes or other destructively acting agencies.

Microparticles are often used as the solid phase and/or as labels. Theterm “microparticles” in the sense of this invention should beunderstood to mean particles which have an approximate diameter of atleast 20 nm and not more than 20 μm, usually between 40 nm and 10 μm,preferably between 0.1 and 10 μm, particularly preferably between 0.1and 5 μm, and quite especially preferably between 0.15 and 2 μm. Themicroparticles can be regularly or irregularly shaped. They can beballs, spheroids or balls with cavities of greater or lesser size. Themicroparticles can consist of organic or inorganic material or of amixture or combination of both. They can consist of a porous ornonporous, swellable or nonswellable material. In principle, themicroparticles can have any density, however particles with a densitywhich is close to the density of water, such as about 0.7 to about 1.5g/ml are preferred. The preferred microparticles are suspensible inaqueous solutions and suspension-stable for as long as possible. Theymay be transparent, partly transparent, or opaque. The microparticlescan consist of several layers, such as for example the so-called“core-and-shell” particles with a core and one or more enclosing layers.The term microparticles for example includes dyestuff crystals, metalsols, silica particles, glass particles, magnetic particles, polymerparticles, oil droplets, lipid particles, dextran and proteinaggregates. Preferred microparticles are particles suspensible inaqueous solutions and consisting of water-insoluble polymeric material,in particular of substituted polyethylenes. Quite especially preferableare latex particles, for example of polystyrene, acrylic acid polymers,methacrylic acid polymers, acrylonitrile polymers,acrylonitrile-butadiene-styrene, polyvinyl acetate-acrylate,polyvinylpyridine or vinyl chloride-acrylate. Of particular interest arelatex particles with reactive groups on their surface such as forexample carboxyl, amino or aldehyde groups, which allow covalent bindingfor example of specific binding partners to the latex particles. Thepreparation of latex particles is for example described in EP 0 080 614,EP 0 227 054 and EP 0 246 446.

A “signal-generating system” can consist of one or more components,where at least one component is a detectable label. A label should beunderstood to mean any molecule which itself produces a signal or caninduce the production of a signal, such as for example a fluorescentsubstance, a radioactive substance, an enzyme or a chemiluminescentsubstance. The signal can for example be detected or measured on thebasis of enzyme activity, luminescence, light absorption, lightscattering, emitted electromagnetic or radioactive radiation or achemical reaction.

A label is capable of generating a detectable signal itself, so that noother components are necessary. Many organic molecules absorbultraviolet and visible light, as a result of which these molecules canreach an excited energy state and emit the absorbed energy in the formof light of a wavelength other than that of the irradiation light.Again, other labels can create a detectable signal directly, for exampleradioactive isotopes or dyes.

Again, other labels require other components for signal creation, i.e.the signal-producing system in such a case includes all the componentsneeded for generation of the signal, such as for example substrates,coenzymes, quenchers, accelerators, additional enzymes, substances whichreact with enzyme products, catalysts, activators, cofactors,inhibitors, ions, etc.

Examples of suitable labels are enzymes including horseradishperoxidase, alkaline phosphatase, glucose-6-phosphate dehydrogenase,alcohol dehydrogenase, glucose oxidase, β-galactosidase, luciferase,urease and acetylcholinesterase; dyes; fluorescent substances includingfluorescein isothiocyanate, rhodamine, phyco-erythrin, phycocyanin,ethidium bromide, 5-dimethyl-aminonaphthalen-1-sulfonyl chloride andfluorescent chelates of rare earths; chemiluminescent substancesincluding luminol, isoluminol, acridinium compounds, olefins, enolethers, enamine, aryl vinyl ethers, dioxene, arylimidazole, lucigenin,luciferin and aequorin; sensitizers including eosin,9,10-dibromo-anthracene, methylene blue, porphyrin, phthalocyanine,chlorophyll, rose Bengal; coenzymes; enzyme substrates; radioactiveisotopes including 125I, 131I, 14C, 3H, 32P, 33P, 35S, 51Cr, 59Fe, 57Coand 75Se; particles including magnetic particles or particles,preferably latex particles, which can themselves be labeled for examplewith dyes, sensitizers, fluorescent substances, chemi-luminescentsubstances, isotopes or other detectable labels; sol particles includinggold or silver sols; liposomes or cells, which can themselves be labeledwith detectable labels; etc. [see also EP-A2-0 515 194; U.S. Pat. No.5,340,716; U.S. Pat. No. 5,545,834; Bailey et al. (1987) J.Pharmaceutical & Biomedical Analysis 5: 649-658].

A signal-generating system can also include components which can enterinto a detectable interaction when in spatial proximity to one another,for example in the form of energy donors and energy acceptors such asfor example photosensitizers and chemiluminescent substances (EP-A2-0515 194), photosensitizers and fluorophores (WO 95/06877), radioactiveiodine125 and fluorophores [Udenfriend et al. (1985) Proc. Natl. Acad.Sci. 82: 8672-8676], fluorophores and fluorophores [Mathis (1993) Clin.Chem. 39: 1953-1959] or fluorophores and fluorescence-quenchers (U.S.Pat. No. 3,996,345).

Interaction between the components includes the direct transfer ofenergy between the components, for example by light or electronradiation or via short-lived reactive chemical molecules. Also includedtherein are processes wherein the activity of one component is inhibitedor intensified by one or more others, for example the inhibition orintensification of enzyme activity or the inhibition, intensification ormodification (for example wavelength shift, polarization) of theelectromagnetic radiation emitted by the component affected. Interactionbetween the components also includes enzyme cascades. In this case, thecomponents are enzymes, whereof at least one provides the substrate foranother, so that a maximal or minimal reaction rate of the coupledsubstrate conversion results.

An effective interaction between the components as a rule takes placewhen these are in spatial proximity, i.e. for example within a distancerange of a few μm, in particular within a distance range of below 600nm, preferably below 400 nm, quite especially preferably below 200 nm.

The term “associated” should be broadly understood and includes forexample a covalent and a noncovalent bond, a direct and an indirectbond, adsorption onto a surface and inclusion into a depression or acavity, etc. With a covalent bond, the antibodies or binding partnersare bound to the solid phase or to the label via a chemical bond.Examples of a noncovalent bond are surface adsorption, inclusion intocavities or the binding of two specific binding partners. As well asdirect bonding to the solid phase or the label, the antibodies orbinding partners can also be bound to the solid phase or the labelindirectly via specific interaction with other specific binding partners(see also EP-A2-0 411 945). Examples of this are: biotinylatedantibodies which can be bound to the label via label-bound avidin or afluorescein-antibody conjugate which can be bound to the solid phase viasolid phase-bound anti-fluorescein antibody or an antibody which can bebound to the solid phase or the label via immunoglobulin-bindingproteins.

A further object of this invention are antibodies or specific bindingpartners according to the invention which are used as in vitrodiagnostic agents or as a component of an in vitro diagnostic agent.

With an in vitro diagnostic agent, the analyte to be detected, forexample a particular PIGF form, is detected or the concentration orcontent thereof determined in a sample outside a living human or animalbody.

A “sample” in the sense of the invention should be understood to meanthe material which probably contains the substance to be detected (forexamples of these, see EP-A2-0 515 194, “Analyt”). The term sampleincludes for example biological fluids or tissue in particular of manand animals, such as blood, plasma, serum, sputum, exudate,bronchoalveolar lavage, lymphatic fluid, synovial fluid, seminal fluid,vaginal mucus, feces, urine, cerebrospinal liquor, hair, skin, tissuesamples or sections. Also included are cell culture samples, plantfluids or tissue, forensic samples, water and effluent samples,foodstuffs and medicaments. If necessary, the samples must be pretreatedin order to make the analyte accessible to the detection method or inorder to remove interfering sample components. Such pretreatment ofsamples may include the separation and/or lysis of cells, theprecipitation, hydrolysis or denaturation of sample components such asfor example proteins, centrifugation of samples, treatment of the samplewith organic solvents such as for example alcohols, in particularmethanol, or treatment of the sample with detergents. Commonly, thesample is transferred into another, most often aqueous, medium whichshould as far as possible not interfere with the detection process.

The antibodies according to the invention can be used in a process forthe quantitative or qualitative determination of an analyte, preferablyparticular PIGF forms, in particular fPIGF, in a sample.

In a quantitative determination, the content, the concentration or theactivity (for example enzyme activity) of the analyte in the sample ismeasured. The term “quantitative determination” also includessemi-quantitative methods which only ascertain the approximate content,concentration or activity of the analyte in the sample or can only beused to give a relative content, concentration or activity value.Qualitative determination should be understood to mean the detection ofthe presence of the analyte in the sample at all, or the demonstrationthat the concentration or activity of the analyte in the sample liesabove or below a defined or several defined threshold values.

The invention thus also relates to methods for the quantitative orqualitative determination of an analyte, preferably particular PIGFforms, in particular fPIGF, in a sample and suitable reagents for this.

For the determination of analytes, binding tests are commonly usedwherein a conclusion can be reached as to the presence, absence orcontent of the analyte in a sample by a specific binding of the analyteto be determined to analyte-specific binding partners. Immunoassays oralso methods wherein oligo- or polynucleotides are hybridized areexamples of binding tests.

The so-called “heterogenous binding tests” are characterized by one ormore separation steps and/or washing steps. The separation can forexample be effected by immune precipitation, precipitation withsubstances such as polyethylene glycol or ammonium sulfate, filtration,magnetic separation, or binding to a solid phase. With heterogenousbinding tests in the sandwich format, as a rule one of theanalyte-specific binding partners is bound to a solid phase and servesfor the removal of the binding complex “analyte/analyte-specific bindingpartner” from the liquid phase, while the other analyte-specific bindingpartner bears a detectable label, for example an enzyme, a fluorescentor chemiluminescent label, etc., for the detection of the bindingcomplex. These test methods are further subdivided into so-calledone-step sandwich tests, wherein the two specific binding partners aresimultaneously incubated with the sample, and two-step sandwich tests,wherein the sample is first incubated with the solid phase reagent andafter a separation and washing step the solid phase-bound bindingcomplex of analyte and analyte-specific binding partners is incubatedwith the detection reagent.

In “homogenous binding tests” no separation is effected between freecomponents of the signal-generating system and those bound to the“analyte/analyte-specific binding partner” complex. The test mixturewhich contains the analyte-specific binding partner, thesignal-generating components and the sample, is assayed after or evenduring the binding reaction without a further separation and/or washingstep and the corresponding measurement signal is determined. Examples ofhomogenous immunoassays [see also Boguslaski & Li (1982) AppliedBiochemistry and Biotechnology 7: 401-414] are turbidimetric ornephelometric methods, wherein the analyte-specific binding partnersused for the determination can be linked to latex particles, such as forexample EMIT® tests; CEDIA® tests; fluorescence-polarizationimmuno-assays; luminescent oxygen channeling immunoassays [LOCI®, seeEP-A2-0 515 194; Ullman et al. (1994) Proc. Natl. Acad. Sci. 91:5426-5430; Ullman et al. (1996) Clinical Chemistry 42: 1518-1526] etc.In a homogenous sandwich immunoassay, such as for example anephelometric latex test, the antibody reagents are incubated togetherwith the sample, and the measurement of the signal is performed duringand/or after the incubation, without a separation or washing step beingperformed before the measurement. In other words: no separation of theantibody-bound analyte from the free analyte or from antibodies whichhave not bound to any analyte is carried out.

Homogenous and heterogenous binding tests can also be carried out in theform of a so-called “sandwich assay”. In this, for example in aheterogenous binding test, the analyte is bound by one solidphase-associated, analyte-specific binding partner and oneanalyte-specific binding partner, which is linked to a component of asignal-generating system. In sandwich immunoassays, antibodies orantigens or haptens can constitute the analyte-specific bindingpartners.

A further specific embodiment of a heterogenous or homogenous bindingtest is the “indirect immunoassay”. In this case, the analyte is anantibody. One of the analyte-specific binding partners is the antigen orfor example a peptide according to the invention or a modified antigenof the antibody to be determined (=analyte), and the otheranalyte-specific binding partner is as a rule an immunoglobulin-bindingprotein such as for example an antibody, which can specifically bind tothe antibodies to be determined (=analyte).

In a homogenous or heterogenous “competitive binding test”sample-analyte and reagent-analyte compete for binding to a limitednumber of analyte-specific binding partners. The reagent-analyte is forexample a “modified analyte”, such as for example a labeled or markedanalyte, an analyte fragment such as for example the peptides accordingto the invention or an analyte analog. Examples for illustration of theprinciple: (i) sample-analyte competes with reagent-analyte, which islinked to a component of a signal-generating system, for binding tosolid phase-linked, analyte-specific binding partners or (ii)sample-analyte competes with solid phase-linked analyte(=reagent-analyte) for binding to analyte-specific binding partnerswhich are linked to a component of a signal-generating system.

The detection of different PIGF forms with the specific binding partnersaccording to the invention, in particular antibodies, can also beeffected by methods such as for example Western blot, dot blot, immunoelectrophoresis, immunofixation electrophoresis, electroimmunodiffusion,immunoprecipitation, radial immunodiffusion, immunofixation,immunochromatography, latex agglutination, turbidimetric ornephelometric test, homogenous or heterogenous binding test, one ortwo-step test, sandwich test, indirect test, competitive test,“point-of-care” test, etc. These and other detection methods are forexample described in “Labor and Diagnose”, ed. L. Thomas, TH-BooksVerlagsgesellschaft mbH, Frankfurt, 1998, Chapter 60 or in “LaboratoryTechniques in Biochemistry and Molecular Biology—An Introduction toRadioimmunoassay and Related Techniques”, ed. T. Chard, Elsevier,Amsterdam, 1987.

The term “point-of-care tests” or “POC tests” includes tests wherein noseparate analytical or measuring instrument is needed for theimplementation or assessment of the test. POC tests are in many casesbased on immunochromatographic methods, immune complex separations byfiltration and/or immunofixation techniques. The POC tests areparticularly intended for measurements on site, for example at thesickbed or at home, for the emergency doctor and/or the private doctorand less for the large laboratory. POC tests can in particular also beperformed by persons who do not have detailed medical and technicaltraining and experience in the field of laboratory medicine. The term“POC tests” in the sense of this invention should also be understood tomean so-called home tests or OTC tests, which can be performed bymedical laymen, such as for example the various pregnancy tests whichare marketed for home use. Other POC tests for example relate to thedetection of cardiac infarction markers, drugs, medicaments, andinfection and inflammation markers. In many POC tests, specific bindingpartners are or in the course of the test become bound to or onto filteror chromatography strips. A positive or negative detection reaction canfor example be coupled with the appearance or nonappearance of a coloredband in a defined test field and/or the appearance or nonappearance of acertain symbol, for example the appearance or nonappearance of a “+” ora “−” and/or the intensity of the particular measurement signal.

A POC test for particular PIGF forms, in particular fPIGF, can forexample be structured as follows: sample and labeled specific antibodieswhich can bind to the fPIGF form, but not or hardly at all to other PIGFforms, are applied onto a test strip. Examples of suitable labels aredyed latex particles, colloidal gold, enzymes etc. If the fPIGF form iscontained in the sample, fPIGF/antibody complexes will form. Thesecomplexes move, for example by capillary forces, in the direction of aregion wherein other specific binding partners, in particularantibodies, which can bind to other fPIGF epitopes and which areimmobilized for example in the form of a band or become immobilized inthe course of the test procedure (for example via a biotin-avidinbridge). The labeled fPIGF/antibody complexes become bound in thisregion and form a sandwich complex with the immobilized specific bindingpartners, in particular antibodies. The intensity of the label signalhere is proportional to the fPIGF sample concentration. In a competitivePOC test method, for example antibody fragments can be immobilized in aregion of the test strip, or become immobilized in the course of thetest procedure. This immobilized antibody would compete with the fPIGFform from the sample for binding to labeled anti-fPIGF antibodies.Alternatively, immobilized fPIGF antibodies and labeled fPIGF protein orthe peptides according to the invention can also be used for theconstruction of a competitive fPIGF test.

A particularly preferable embodiment of the method according to theinvention is a nephelometric or turbidimetric test, in particular such atest wherein antibodies according to the invention—preferably linked tomicroparticles (in particular to latex particles)—are used.

Another object according to the invention is a test kit which containsone or more of the antibodies and/or peptides according to theinvention. Such a kit normally contains all or only some components of atest in packaged form. The antibodies and/or peptides according to theinvention can for example be linked to one or more solid phases and/orone or more components of a signal-generating system. The test kit canfor example contain standards, controls and other reagents, such as forexample buffers, washing solutions, measurement signal-triggeringsolutions and/or enzyme substrate, cuvettes, pipettes and/or testdirections. A particularly preferable test kit according to theinvention contains antibodies according to the invention and/or peptidesaccording to the invention linked to latex particles.

The antibodies and peptides according to the invention can also be usedfor affinity chromatography. The term “affinity chromatography” shouldbe understood to mean a method for the purification and isolation ofsubstances, in particular biopolymers, which is based on the fact thatmany substances can enter into a selective, noncovalent, reversible bondwith binding partners specific to them. The principle of the methodconsists in that the specific binding partner is as a rule covalentlybound to an insoluble matrix (for example porous glasses, or agarose,cellulose, dextran, polymer and silica gel-based gels) and placed incontact with a sample containing the substance. The test substance isimmobilized and retained on account of its specific interaction with thematrix-bound specific binding partner, while all other substancescontained in the sample are removed by elution. The test substance isthen released from the matrix with a suitable elution agent whicheliminates the noncovalent bonding between substance and specificbinding partner (see also E. Buddecke, 1989, Grundrisse der Biochemie,Walter de Gruyter, Chapter 7 “Proteine”).

A further object of this invention are antibodies or specific bindingpartners according to the invention which are used as therapeuticagents. This includes antibodies according to the invention or peptidesaccording to the invention in a pharmaceutically compatible, sterileinjection medium. A pharmaceutically compatible, sterile injectionmedium should for example be understood to mean a germ-free,pyrogen-free solution, for example saline or another electrolytesolution, such as is normally used for intravenous, intramuscular,intraperitoneal or subcutaneous administration of drugs, vaccines orcontrast media.

A further object of this invention is the use of the antibodiesaccording to the invention as diagnostic agents or as a component of adiagnostic agent.

A further object of this invention is a process for the preparation ofan antibody according to the invention, which is characterized in thatone or more of the peptides described above are used for theimmunization.

The antibodies according to the invention can also be produced by theuse of naturally occurring and/or recombinant PIGF and VEGF proteins,protein isoforms or fragments thereof.

A further object of the invention is the use of proteins, proteinisoforms, fragments, degradation products, homologs, and peptidesaccording to the invention as reference materials, standards,calibrators and controls. Reference materials are materials orsubstances which have properties which are established in such a mannerthat reference materials are used as calibrators, standards andcontrols. In addition, reference materials can be used for thevalidation of measurement methods and the assignment of defined values,in particular “conventionally correct values”. The use of referencematerials as calibrators, standards and controls or the use ofcalibrators, standards and controls which relate to reference materialsor to the “conventional correct values” stated there is important inquality control and quality assurance.

The peptides used as the immunization antigen can be used for theimmunization unbound and/or carrier-bound.

Typical carriers are for example proteins, such as for exampleovalbumin, albumin or keyhole limpet hemocyanin (KLH), or polymers, suchas for example polyethylene glycol, polyacrylamide orpoly-d-glutamine-d-lysine. The peptides can for example be bound tothese carriers by means of carbodiimide or glutaraldehyde or also bymeans of a heterobifunctional reagent, which can also act as a spacer,such as for example N-maleimido-butyryloxysuccinimide ester (GBMS). Forother examples and coupling methods, see also Wong, S. (1993) Chemistryof Protein Conjugation and Cross-Linking, CRC Press, Inc., Boca Raton.

The immunization antigen can for example be dissolved inphosphate-buffered saline and treated with Immun Easy Mouse adjuvant.This emulsion can then be administered for example intradermally,intraperitoneally and/or subcutaneously to an animal, for example arabbit, mouse, rat, guinea pig, horse, donkey, sheep, goat, chicken,etc. Booster injections, wherein the immunization antigen can also beemulsified with incomplete Freund adjuvant, can help to intensify theimmune response.

Polyclonal antibodies according to the invention can be obtained fromthe antiserum of the immunized animals and can be further purified byaffinity chromatography over a matrix, to which for example the relevantPIGF forms or the peptides used as the immunization antigen can bebound.

In order to create monoclonal antibodies according to the invention, theimmune cells of immunized animals, such as for example of a mouse orrabbit, are fused with myeloma cells to create antibody-producinghybridoma cells and then suitable clones are isolated by the generallyknown procedure [see also Harlow & Lane (1988) Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor; Peters et al.(1985) Monoklonale Antikörper: Herstellung and Charakterisierung,Springer Verlag]. The selection of the clones producing the desiredmonoclonal antibodies is carried out by means of specific screeningmethods. In these, the binding specificity of the antibodies releasedinto the cell culture supernatant, for example to the immunizationantigen or to some carrier of the immunization antigen, is checked byenzyme immunoassay, radioimmunoassay and/or western blotting. Hybridomaswhich produce antibodies according to the invention are reproduced bycloning. The hybridoma cell lines thus obtained are then available forthe permanent production of monoclonal antibodies. Larger quantities ofantibodies can for example be obtained from cell culture supernatant, inparticular from fermenters or roller cultures and from ascites.

Depending on the desired use purpose, it is advantageous to use onlyparts of the antibodies, such as for example Fab-, F(ab′)2-, orFab′-fragments. These can for example be created by the enzymaticcleavage method known to the person skilled in the art [see also Harlow& Lane (1988) Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, Cold Spring Harbor].

The antigen binding sites of an antibody are situated in the so-calledvariable domains, which are encoded by the V genes. Thus with the knowngenetic engineering methods [for example Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, 2nd edition; McCafferty et al. (1990) Nature 348:552-554] the corresponding nucleic acid sequence of an antibodyaccording to the invention can also be determined and via this also thecorresponding amino acid sequence, if this was not already known fromamino acid sequencing. As the starting material for such analyses, thehybridoma cells or the antibody-producing immune cells of immunizedanimals can be used.

With the knowledge of the nucleic acid and/or amino acid sequence, it isthen possible by means of standard genetic engineering and molecularbiological methods [see also Johnson & Chiswell (1993) Current Opinionin Structural Biology 3: 564-571] to prepare humanized, chimeric, bi- oroligospecific antibodies, and also peptides derived from the“complementarity determining region” (“minimal recognition units”),single-chain fragments, and/or functional fusion products, for examplerecombinantly produced antibody-enzyme constructs [see also Larrick &Fry (1991) Human Antibodies and Hybridomas 2: 172-189; Kitano et al.(1986) Appl. Microbiol. Biotechnol. 24: 282-286; Thompson et al. (1986)J. Immunol. Methods 94: 7-12], which bind to the particular, specificepitopes of the PIGF forms, in particular to a peptide according to theinvention. With such peptides included under the term “antibodies”, forexample a reduction in the immunogenicity and/or intensified activity onadministration as a medicament or as an in vivo diagnostic agent can beachieved and/or advantages result for use as or in an in vitrodiagnostic agent. The antibodies are also preparable, if necessary withrecourse to genetic engineering methods, in fungi such as for exampleyeast cells [Fischer et al. (1999) Biol. Chem. 380: 825-839; Hiatt etal. (1992) Genetic Engineering 14: 49-64), plant, animal and prokaryoticcells (see also WO 95/25172) and isolated human cells.

A further object of this invention are also fungi, animal, plant orprokaryotic cells and isolated human cells which produce an antibodyaccording to the invention. A preferred embodiment of this inventionincludes hybridoma cell lines which produce the antibodies according tothe invention.

The examples described below serve for the illustration of individualaspects of this invention by way of example.

Example 1 Preparation of fPIGF-Specific Monoclonal Antibodies

a) Immunization of Mice

BALB/c mice are each immunized intraperitoneally with 20 μg ofimmunization antigen (peptide with LRCTGCCGDENLHCVPVET (IAN 2) (SEQ IDNO: 26) bound to KLH) in Immun Easy Mouse adjuvant (Qiagen GmbH,Germany). IAN 2 was synthesized by solid phase synthesis according togenerally known methods. After 4 and 8 weeks, a booster injection with20 μg each of immunization antigen without adjuvant was performed. Inthe last 3 days before the fusion, the mice were intravenously boostedwith 10 μg of immunization antigen each.

b) Fusion

After sacrifice of the mice by CO₂ inhalation, the spleens are removedand single cell suspensions in serum-free Dulbecco's modified EagleMedium (DMEM; PAN Biotech GmbH, Germany) are prepared. The cells arecentrifuged (652×g) and washed twice in DMEM. Next the cell count isdetermined by Trypan blue staining. 2×10⁷ myeloma cells (Sp2/0) areadded for about 10⁸ spleen cells. After centrifugation (360×g) thesupernatant is discarded, 1 ml of polyethylene glycol solution (PEG4000, Merck Eurolab GmbH, Germany; ca. 50% in DMEM) is added to the cellpellet and incubated for 1 minute at 37° C. after resuspension. Next ca.10 ml of DMEM are added, and the mixture incubated for 2 to 4 minutes atroom temperature. The fused cells are centrifuged down (326×g) and thepellet is resuspended in DMEM+10% fetal calf serum (Bio WhittakerEurope, Belgium)+HAT medium (CC Pro GmbH, Germany) and filled into24-well cell culture plates (Corning Costar GmbH, Germany). Theapproximate cell concentration is 5×10⁴ to 5×10⁶ cells per well.

After 2 to 3 weeks, the cell colonies formed (hybrids) are removed andtransferred to new culture plates.

c) Screening

The specificity of the antibodies released into the cell culture istested in a first test step by means of microtiter plates (Nunc GmbH &Co. KG, Germany) which are coated with a peptide with the amino acidsequence LRCTGCCGDENLHCVPVET (SEQ ID NO: 26).

100 μl of cell culture supernatant (dilution 1:2) are pipetted into eachwell of the microtiter plate and incubated for 1 hour at +15 to +25° C.The plates are washed twice with washing solution POD (OSEW; DadeBehring Marburg GmbH, Germany) and then 100 μl of anti-mouse IgG/F(ab′)2POD conjugate (Dade Behring Marburg GmbH, Germany) are filled into eachwell and incubated for 1 hour at +15 to +25° C. After two furtherwashings of the plate, 100 μl of Chromogen TMB-solution (Dade BehringMarburg GmbH, Germany) are filled into each well and incubated for afurther 30 minutes at +15 to +25° C. After the incubation, 100 μl ofstop solution POD (Dade Behring Marburg GmbH, Germany) are filled intoeach well and the microtiter plate is assessed at 450 nm on the BEP II(Behring-ELISA Processor II, Dade Behring Marburg GmbH, Germany).

In a second test step, the hybrids are tested again in the same testformat after isolation as described above.

d) Cloning

Individual cells of hybrids which produce fPIGF-specific antibodies arecloned with a micromanipulator (Leitz Messtechnik GmbH, Germany).Culture supernatants of these clones are purified as described in g) andcharacterized in more detail as described in e), h) and i).

e) Determination of Antibody Class

The subclass of the antibodies against fPIGF is determined using theIsoStrip™ Mouse Monoclonal Antibody Isotyping Kit from BoehringerMannheim Co., Germany.

f) Production of Antibodies

For the production of large quantities of antibodies, the relevant cellclones are transferred to roller bottles (Corning Costar GmbH, Germany)and expanded to the desired final volume at +37° C. Subsequently, theroller culture suspension is filtered through 0.22 μm to remove thecells. The now cell-free antibody solution is concentrated byUltrafilter (separation limit 30,000 Dalton) and then purified.

g) Purification of the Antibodies

The antibody solution obtained is rebuffered with 0.14 M phosphatebuffer pH 8.6 and applied onto a chromatography column filled withrProtein A Sepharose™ Fast Flow (Amersham Biosciences Europe GmbH,Germany) (1 ml of rProtein A Sepharose™ Fast Flow is used per 10 mg ofantibody to be purified). All non-bound components are removed bywashing the column with 0.14 M phosphate buffer pH 8.6. The boundantibody is eluted from the column with 0.1 M citric acid pH 3.0 anddialyzed against 0.05 M sodium acetate+0.5 M NaCl+0.05 M Tris+0.01%sodium azide pH 7.0.

h) Selection of Suitable Antibodies for an fPIGF Sandwich ELISA

The reaction of the monoclonal anti-fPIGF antibodies with thefPIGF-specific epitope (for example the peptide with the amino acidsequence LRCTGCCGDENLHCVPVET (SEQ ID NO: 26)) is investigated:

Reaction with fPIGF:

As the solid phase, a microtiter plate which is coated with fPIGF isused. The anti-fPIGF antibodies from culture supernatants are incubatedon this. After a washing step, binding of the antibody to the PIGF isdetected via a conjugate consisting of polyclonal anti-mouse antibodiesfrom the rabbit and the enzyme peroxidase with subsequent colorreaction.

Reaction with an sFlt-1/PIGF Complex:

As the solid phase, a microtiter plate which is coated with fPIGF isused. sFlt-1 is incubated in this. The sFlt-1 which is used for this is“recombinant human VEGF R1 (Flt-1)/Fc Chimera” from R&D Systems (Catalognumber: 321-FL or 321-FL/CF). After a washing step, anti-fPIGFantibodies from culture supernatants are incubated. After a washingstep, no or only slight binding of specific anti-f PIGF antibodies willbe detectable, since the binding sites are mainly occupied by sFlt-1.fPIGF-nonspecific antibodies also bind more to the sFlt-1/PIGF complex,i.e. gPIGF. After a washing step, binding of this nonspecific antibodyis detected by a conjugate consisting of polyclonal anti-mouseantibodies from the rabbit and the enzyme peroxidase with subsequentcolor reaction.

In this test system, fPIGF-specific antibodies are those which show noor a markedly lesser color reaction in the reaction with an sFlt-1/PIGFcomplex than in the reaction with a fPIGF-specific peptide.

Accordingly, fPIGF-specific antibodies are selected. The suitability ofthese antibodies for use as solid phase antibodies in a sandwich ELISAwith fPIGF-specific conjugate antibodies which is coupled to horseradishperoxidase by a procedure known to the person skilled in the art (forexample Nakane conjugation) is investigated.

The suitability is checked in the sandwich ELISA as described in Example2a). The essential decision criteria for suitability are a cleardifferentiation between fPIGF and sFlt-1/PIGF complex. Further criteriaare the lower detection limit and the linearity of the calibrationcurve.

Example 2 Preparation of fPIGF-Specific Monoclonal Antibodies

a) Immunization of Mice

BALB/c mice are each intraperitoneally immunized with 20 μg ofimmunization antigen (peptide with GCCGDENLHK (IAN 2-1-1K) (SEQ ID NO:29) bound to KLH) in Immun Easy Mouse adjuvant (Qiagen GmbH, Germany).IAN 2-1-1K was synthesized by solid phase synthesis according togenerally known methods. The purity of IAN 2-1-1K was checked by columnchromatography (column: Merck 250×4 mm). For this, 0.1% TFA/water wasused as buffer A, and 0.08% FFA/acetonitrile as buffer B. The flow ratewas 0.8 ml. Detection was effected at 220 nm. Further, matrix-assistedlaser desorption ionization mass spectrometry (MALDI-MS) was performed.The main peak was at 1075 m/z. After 4 and 8 weeks, a booster injectionwith 20 μg each of immunization antigen without adjuvant was performed.The last 3 days before the fusion, the mice are boosted with 10 μg eachof immunization antigen.

The fusion b), cloning d), determination of antibody class e),production of antibodies f), purification of antibodies g) and selectionof suitable antibodies for an fPIGF sandwich ELISA h) are performed asin Example 1. For the screening c), the same procedure as in Example 1is also followed, with the exception that the specificity of theantibodies released into the cell culture is tested in a first test stepby means of microtiter plates (Nunc GmbH & Co. KG, Germany) which arecoated with a peptide with the amino acid sequence GCCGDENLHK (IAN:2-1-1K) (SEQ ID NO: 29).

Example 3 Detection of fPIGF in a Sample

a) Test Method A

Peroxidase-conjugated anti-PIGF antibodies are used in combination witha monoclonal anti-fPIGF antibody according to the invention in an enzymeimmunoassay in accordance with the sandwich principle.

During the first incubation, the fPIGF contained in the sample—ifpresent—binds to the antibodies according to the invention directedagainst fPIGF, which are immobilized on the surface of the wells of amicrotitration plate. The wells are rinsed out and thenperoxidase-conjugated anti-PIGF antibodies which are directed againstany epitope—except for the receptor-binding epitope—of PIGF are used ina second binding reaction. With the aid of specificperoxidase-conjugated anti-PIGF antibodies, in principle the presence ofparticular PIGF isoforms can also be detected in this test method. Forexample, a PIGF-1-specific antibody can be used for the detection ofPIGF-1, etc. In principle, the detection of the presence of differentPIGF isoforms is possible with different specific antibodies. The excessenzyme-conjugated antibodies are washed off. Subsequently, the boundenzyme activity in the wells is determined. The enzymatic reaction ofhydrogen peroxide and tetramethylbenzidine is stopped by addition ofdilute sulfuric acid. The color intensity proportional to the fPIGFantigen concentration is determined photometrically at a wavelength of450 nm and either qualitatively assessed by means of a cut-off orquantified on the basis of a calibration curve based on standards.

Such a sandwich immunoassay according to the invention detects fPIGFspecifically in just one test procedure.

b) Test Method B

Peroxidase-conjugated, monoclonal anti-fPIGF antibodies according to theinvention are used in combination with a monoclonal anti-fPIGF antibodyaccording to the invention in an enzyme immunoassay according to thesandwich principle.

As in test method A, during the first incubation, the fPIGF contained inthe sample—if present—binds to the antibodies according to the inventiondirected against fPIGF, which are immobilized on the surface of thewells of a microtitration plate. The wells are rinsed and thenperoxidase-conjugated anti-fPIGF antibodies are used in a second bindingreaction. The excess enzyme-conjugated antibodies are washed out. Next,the bound enzyme activity in the wells is determined. The enzymaticreaction of hydrogen peroxide and tetramethylbenzidine is stopped byaddition of dilute sulfuric acid. The color intensity proportional tothe fPIGF antigen concentration is determined photometrically at awavelength of 450 nm and either qualitatively assessed by means of acut-off or quantified on the basis of a calibration curve based onstandards.

Such a sandwich immunoassay according to the invention detects fPIGFspecifically in just one test procedure. Test method B is particularlypreferred since fPIGF is generally present as a homodimer, for exampleas fPIGF-1 homodimer, and receptor can be bound at both poles of thehomodimer. In test method B, an enzyme immunoassay according to thesandwich principle is used which utilizes one immobilized anti-fPIGFantibody and one peroxidase-conjugated anti-fPIGF antibody, so thatfPIGF which has no binding partner at both poles is predominantlydetermined.

According to the examples and test methods described, the specificitiesof the other binding partners according to the invention, in particularof the antibodies, can also analogously be determined and useddiagnostically.

Example 4 Preparation of Other fPIGF-Specific Monoclonal Antibodies

The following monoclonal antibodies were selected and investigated fortheir recognition of free and bound PIGF forms in accordance withExample 1. For the immunization, the immunization antigens stated in thefollowing table were used. The antibody “MAB264” from R&D Systems is anantibody from the state of the art.

TABLE 1 Monoclonal antibody Immunization Antigen Specificity 05-54/04recombinant human free PIGF 05-64/026 PIGF; 05-81-05R&D Systems, Catalog 05-81-010 No. 264-PG/CF (Group 1) 05-61/016recombinant human free and bound 05-63/020 PIGF; PIGF (ComparisonR&D Systems, Catalog antibodies) No: 264-PG/CF 05-164/012 IAN 3-3-1free PIGF 05-164/038 (RSGDRPSYVELT) 05-164/042 (SEQ ID NO: 37)05-164/054 (Group 2) 05-121/06 IAN 1-1-2 free PIGF 05-120/03(VVPFQEVWG RSY) (Group 3) (SEQ ID NO: 11) R&D Systems free and boundMAB264 PIGF (Comparison antibody, state of the art)

The cell cultures which produce the monoclonal antibodies according tothe invention stated in the table or which produce the comparisonantibodies were deposited at the DSMZ (Deutsche Sammlung vonMikroorganismen and Zellkulturen GmbH), Mascheroder Weg 1b, 38124Braunschweig, German Federal Republic, in accordance with the BudapestTreaty under the following entry numbers allocated by the internationaldeposition office:

Cell culture 2005-81-05=DSM ACC2764

Cell culture 2005-81-010=DSM ACC2765

Cell culture 2005-64/026=DSM ACC2766

Cell culture 2005-63/020=DSM ACC2767

Cell culture 2005-61/016=DSM ACC2768

Cell culture 2005-54/04=DSM ACC2769

Cell culture 2005-164/054=DSM ACC2770

Cell culture 2005-164/042=DSM ACC2771

Cell culture 2005-164/038=DSM ACC2772

Cell culture 2005-164/012=DSM ACC2773

Cell culture 2005-121/06=DSM ACC2774

Cell culture 2005-120/03=DSM ACC2775

For all of the aforesaid cell cultures, the deposition date is 23 Mar.2006.

Example 5 Reactivity of the Antibodies According to the Invention andthe Comparison Antibodies with Recombinant (Free) PIGF

Microtitration plates (Nunc, Type B), were coated with polyclonalantibody against mouse IgG/F(ab)2 (Dade Behring Marburg GmbH, Germany);coating concentration 10 μg/ml≈1.5 μg/well.

100 μl of the monoclonal antibodies to be investigated were pipettedinto the wells of the microtitration plate at a concentration of a) 1.0μg/ml or b) 0.1 μg/ml and incubated for 1 hour at +15 to +25° C. Afterwashing of the microtitration plate three times with washing solutionPOD (Product No.: OSEW; Dade Behring Marburg, Germany) 100 μl of asolution of recombinant PIGF (R&D Systems, Catalog No.: 264-PG/CF) wereadded to each well at a concentration of 0.1 μg/ml and incubated for 1hour at +15 to +25° C. After washing of the microtitration plate threetimes with washing solution POD, 100 μl of anti-human PIGF-POD conjugate(produced by a conventional conjugation method from “Human PIGF AffinityPurified Polyclonal Antibody”, Prod. No.: AF-264-PB/R&D Systems) werefilled into each well and incubated for 1 hour at +15 to +25° C. After afurther washing of the microtitration plate three times, 100 μl ofChromogen TMB solution (Product No.: OUVF, Dade Behring Marburg GmbH,Germany) were filled into each well and incubated for a further 30minutes at +15 to +25° C. After the incubation, 100 μl of stop solutionPOD (Product No.: OSFA, Dade Behring Marburg GmbH, Germany) were filledinto each well and the microtitration plate was assessed at 450 nm onthe BEP II (Dade Behring Marburg GmbH, Germany). The results are listedin Table 2.

TABLE 2 Determination of reactivity with PIGF by assessment of themicrotitration plates at 450 nm on the BEP II. Extinction at 450 nmAntibody Antibody according according to to state of Antibody accordingto invention, Antibody according to invention, invention, Comparison theart Antibody Group 1 Group 2 Group 3 antibody R&D concentration 05-54/05-64/ 05-81- 05-81- 05-164/ 05-164/ 05-164/ 05-164/ 05-121/ 05-61/05-63/ Systems: [μg/ml] 04 026 05 010 012 038 042 054 06 016 020 MAB2641.0 2.5 2.5 2.5 2.5 0.323 0.370 0.389 0.396 1.343 2.5 2.5 2.5 0.1 2.52.073 0.659 0.609 0.099 0.145 0.138 0.126 0.177 2.5 2.5 1.896

Example 6 Reactivity of the Antibodies According to the Invention, theComparison Antibodies and the Antibodies from the State of the Art withPIGF/sFlt-1 Complex (gPIGF)

Microtitration plates (Nunc, Type B) were coated with polyclonalantibodies against human IgG/Fc (Dade Behring Marburg GmbH, Germany).Coating concentration 2.5 μl/ml≈0.376 μg/well.

100 μl of a solution of recombinant human VEGF R1 (Flt-1)/Fc chimera:R&D Systems, Catalog No.: 321-FL/CF) were placed in each well of themicrotitration plate at a concentration of 1 μg/ml and incubated for 1hour at +15 to +25° C. After washing of the microtitration plate threetimes with washing solution POD (see Example 5), for the preparation ofthe PIGF/sFlt-1 complex 100 μl of a solution of recombinant PIGF (seeExample 5) was added to each well at a concentration of 1 μg/ml andincubated for 1 hour at +15 to +25° C. After washing of themicrotitration plate three times with washing solution POD, 100 μl ofthe monoclonal antibody to be investigated were pipetted into each wellat a concentration of a) 1 μg/ml or b) 0.1 μg/ml and incubated for 1hour at +15 to +25° C. After washing of the microtitration plate threetimes with washing solution POD, 100 μl of anti-mouse IgG/F(ab)₂-PODconjugate (Dade Behring Marburg GmbH, Germany) were filled into eachwell and incubated for 1 hour at +15 to +25° C. After further washing ofthe microtitration plate three times, 100 μg of Chromogen TMB solution(see Example 5) were filled into each well and incubated for a further30 minutes at +15 to +25° C. After the incubation, 100 μl of stopsolution POD (see Example 5) were filled into each well and themicrotitration plate was assessed at 450 nm on the BEP II (see Example5). The results are listed in Table 3.

TABLE 3 Determination of reactivity with PIGF/sFlt-1 complex byassessment of the microtitration plates at 450 nm on the BEP II.Extinction at 450 nm Antibody Antibody according according to to stateof Antibody according to invention, Antibody according to invention,invention, Comparison the art Antibody Group 1 Group 2 Group 3 antibodyR&D concentration 05-54/ 05-64/ 05-81- 05-81- 05-164- 05-164/ 05-164/05-164/ 05-121/ 05-61/ 05-63/ Systems: [μg/ml] 04 026 05 010 012 038 042054 06 016 020 MAB264 1.0 0.036 0.032 0.043 0.045 0.027 0.026 0.0290.023 0.028 2.500 2.500 0.691 0.1 0.035 0.028 0.026 0.026 0.022 0.0230.023 0.023 0.028 0.692 0.750 0.217 The antibodies according to theinvention show no reaction with the PIGF/sFlt-1 complex formed, whereasthe comparison antibodies and the antibody from the state of the artshow a clear reaction.

Example 7 Epitope Mapping

Scans of overlapping peptides which were derived from the sequence ofhuman PIGF (13-meric peptides, 11 amino acids overlapping) were preparedby means of the SPOT synthesis technology. The methods are described in:Wenschuh, H. et al. (2000) “Coherent membrane supports for parallelmicrosynthesis and screening of bioactive peptides”, Biopolymers(Peptide Science), 55:188-206. The peptides were synthesized in definedorder (as a “peptide array”) stepwise on cellulose membranes, so thatthey were present covalently coupled to the cellulose membrane. Bindingtests to assess the immunoreactivity of the peptides were performeddirectly on the arrays. The incubation protocol for this was as follows:

equilibration in TBS buffer, pH 8.0

2 hrs blocking buffer, pH 8.0

2 hrs antibody incubation (3 μg/ml) in blocking buffer, pH 8.0

washing with TBS (0.05% Tween20)

2 hrs incubation with anti-mouse IgG-POD in blocking buffer, pH 8.0

3×5 min washing with TBS (0.05% Tween20)

detection by chemiluminescence (Lumi-Imager, Roche Diagnostics)

Results: The two antibodies according to the invention 05-81-05 and05-81-010 from Group 1 react with the following sequence segments:

1. EKMKPERCGDAVP (SEQ ID NO: 62) 2. MKPERCGDAVPRR (SEQ ID NO: 63)The sequence recognized is identical with the C-terminal domain ofPIGF-1.Additional Tests with the Test Monoclonal Antibodies on the Solid Phase:

In the following Examples 8 and 9 only the higher affinity antibodies ofGroup 1 were tested in comparison to the state of the art and comparisonantibodies.

Example 8 Reaction with PIGF

Microtitration plates (see Example 5) were coated with the monoclonalantibodies according to the invention, with the comparison antibodiesand with monoclonal antibodies from the state of the art. Coatingconcentration 3 μg/ml≈0.45 μg/well.

100 μl of a geometric dilution series starting with 50 ng/ml ofrecombinant PIGF (see Example 5) were pipetted into the wells of themicrotitration plate and incubated for 1 hour at +15 to +25° C. Aftertriple washing of the plate with washing solution POD (see Example 5),100 μl of anti-human PIGF-POD conjugate (see Example 5) were filled intoeach well and incubated for 1.5 hours at +15 to +25° C. After furthertriple washing of the plate, 100 μl of Chromogen TMB solution (seeExample 5) were filled into each well and incubated for a further 30minutes at +15 to +25° C. After the incubation, 100 μl of stop solutionPOD (see Example 5) were filled into each well and the microtitrationplate was assessed at 450 nm on the BEP II (see Example 5). The resultsare listed in Table 4.

TABLE 4 Determination of reactivity with PIGF by assessment of themicrotitration plates at 450 nm on the BEP II Extinction at 450 nmAntibody according to Antibody according to invention, Comparison stateof the art Conc. Group 1 antibody R&D Systems: [ng/ml] 05-54/0405-64/026 05-81-05 05-81-010 5-61/016 06-63/020 MAB264 human 50 2.5002.500 1.603 2.074 2.500 2.500 2.500 recombinant 25 2.500 2.500 1.1341.706 2.500 2.500 1.467 PIGF 12.5 2.500 1.926 0.610 1.154 2.500 2.5000.885 6.25 1.737 1.408 0.805 0.752 1.782 2.049 0.468 3.13 1.144 0.8190.394 0.413 1.165 1.330 0.232 1.56 0.763 0.583 0.363 0.255 0.712 0.8180.081 0.78 0.406 0.376 0.234 0.143 0.414 0.456 0.050

Example 9 Reactivity of the Antibodies According to the Invention, theComparison Antibodies and the Antibodies from the State of the Art withPIGF/sFlt-1 Complex (gPIGF)

Microtitration plates (see Example 5) were coated with the monoclonalantibodies according to the invention, with comparison antibodies andwith monoclonal antibodies from the state of the art. Coatingconcentration 3 μg/ml≈0.45 μg/well.

In a reaction vessel, a geometric dilution series starting with 25 ng/mlof recombinant PIGF was prepared. Recombinant human VEGF R1 (Flt-1)/Fcchimera (see Example 6) at a concentration of 400 ng/ml was added toeach dilution and incubated for 1 hour at +15 to +25° C. Next, 100 μlwere pipetted into each of the wells of the microtitration plate andincubated for 1 hour at +15 to +25° C. After washing of themicrotitration plate four times with washing solution POD (see Example5), 100 μl of anti-human VEGF R1-POD conjugate (R&D Systems, Part 891096from Quantikine Human VEGF R1 Immunoassay; DVR100B) were filled intoeach well and incubated for 1 hour at +15 to +25° C. After washing ofthe microtitration plate a further three times, 100 μl of Chromogen TMBsolution (see Example 5) were filled into each well and incubated for afurther 30 minutes at +15 to +25° C. After the incubation 100 μl of stopsolution POD (see Example 5) were filled into each well and themicrotitration plate was assessed at 450 nm on the BEP II (see Example5). The results are listed in Table 5.

TABLE 5 Determination of reactivity with PIGF/sFlt-1 complex byassessment of the microtitration plates at 450 nm on the BEP II.Extinction at 450 nm Antibody according to Concentration Antibodyaccording to invention, Comparison state of the art [μg/ml] Group 1antibody R&D Systems: PIGF VEGF R1 05-54/04 05-64/026 05-81-05 05-81-01005-61/016 05-63/020 MAB264 25 400 0.079 0.143 0.260 0.204 1.772 2.5002.188 12.5 400 0.047 0.099 0.264 0.105 1.642 2.045 1.611 6.25 400 0.0380.071 0.116 0.088 0.936 1.530 0.883 3.13 400 0.04 0.069 0.072 0.0590.477 0.868 0.529 1.56 400 0.037 0.068 0.046 0.049 0.278 0.404 0.2720.78 400 0.038 0.063 0.042 0.045 0.177 0.282 0.146 0.39 400 0.047 0.0730.043 0.049 0.166 0.189 0.094

The antibodies according to the invention recognize the PIGF/sFlt-1complex only very weakly or not at all. They are specific for free PIGF,while the comparison antibodies and the antibodies from the state of theart show clear reactions, i.e. are not specific for free PIGF.

The present invention is also expressly described by the followingpatent claims, without however restricting the invention thereto.

What is claimed is:
 1. An isolated antibody, wherein the antibody isselected from the group consisting of monoclonal antibody 05-164/054,which is produced by a hybridoma cell line deposited under DepositAccession No. DSM ACC2770, monoclonal antibody 05-164/042, which isproduced by a hybridoma cell line deposited under Deposit Accession No.DSM ACC2771, monoclonal antibody 05-164/038, which is produced by ahybridoma cell line deposited under Deposit Accession No. DSM ACC2772,and monoclonal antibody 05-164/012, which is produced by a hybridomacell line deposited under Deposit Accession No. DSM ACC2773.
 2. Abinding assay for detection of human Placental Growth Factor (PIGF) notbound by Flt-1 receptor (free PIGF) in a sample, comprising the stepsof: (a) bringing the antibody of claim 1 into contact with the sample,(b) determining a binding complex comprising free PIGF from the sampleand the antibody qualitatively or quantitatively.
 3. A test kit forimplementation of a binding assay for detection of human PlacentalGrowth Factor (PIGF) not bound by Flt-1 receptor (free PIGF) in asample, wherein the test kit comprises the antibody of claim 1 andreagents for detecting formation of a binding complex comprising freePIGF bound to the antibody.
 4. An isolated antibody fragment of theantibody of claim 1, wherein the antibody fragment specifically binds toa peptide consisting of the amino acid sequence RSGDRPSYVELT (SEQ ID NO:37).
 5. A binding assay for detection of human Placental Growth Factor(PIGF) not bound by Flt-1 receptor (free PIGF) in a sample, comprisingthe steps of: (a) bringing the antibody fragment of claim 4 into contactwith the sample, (b) determining a binding complex comprising free PIGFfrom the sample and the antibody fragment qualitatively orquantitatively.
 6. A test kit for implementation of a binding assay fordetection of human Placental Growth Factor (PIGF) not bound by Flt-1receptor (free PIGF) in a sample, wherein the test kit comprises theantibody fragment of claim 4 and reagents for detecting formation of abinding complex comprising free PIGF bound to the antibody fragment. 7.An isolated hybridoma cell line, wherein the hybridoma cell line isselected from the group consisting of a hybridoma cell line depositedunder Deposit Accession No. DSM ACC2770, a hybridoma cell line depositedunder Deposit Accession No. DSM ACC2771, a hybridoma cell line depositedunder Deposit Accession No. DSM ACC2772, and a hybridoma cell linedeposited under Deposit Accession No. DSM ACC2773.